Alzheimer's disease (AD) is a neurodegenerative process characterized by the deposition of beta- amyloid proteins (A?) in the brain and cerebrovasculature. While AD has always been a concern for our aging Veterans, the propensity for head injuries sustained in combat has placed our Veterans at even greater risk for developing AD than the general population. Mounting evidence now suggests the excessive accumulation of A? in AD is the result of impaired A? clearance from the brain. Lipoprotein receptors within the blood-brain barrier contribute to the brain-to-blood elimination of A?. However, these receptors are vulnerable to proteolysis at the cell surface (i.e., ectodomain shedding), which diminishes their ability to transport ligands, such as A?. One of the ligands closely associated with the lipoprotein receptors is apolipoprotein E (apoE), which exists as three isoforms in humans (apoE2, apoE3, and apoE4). Importantly, possession of the apoE4 allele represents the strongest genetic risk factor for late-onset AD. Recent reports, including our own, indicate A? clearance from the brain is differentially regulate by the type of apoE isoform expressed. Our preliminary studies demonstrate that apoE4 is less adept than the other isoforms in protecting lipoprotein receptors from the shedding process, which results in reduced A? clearance from the brain. At this stage, however, the mechanism by which apoE influences lipoprotein receptor proteolysis has yet to be elucidated. MMP9 is an endopeptidase that can bind and proteolyze (i.e., shedding) lipoprotein receptors. In AD, MMP9 levels are significantly elevated in the brain and periphery compared to control subjects. To this stage there has been little investigation into the relationship between apoE and MMP9, but our preliminary data suggest a key role for apoE in mediating the effect of MMP9 on lipoprotein receptor shedding. In our preliminary studies, cerebral vessels isolated from apoE2 transgenic mice showed reduced lipoprotein receptor shedding compared to apoE4 cerebrovessels following exposure to activated MMP9. In addition, we found that administration of an MMP9 inhibitor to apoE4 transgenic mice reduced lipoprotein receptor shedding in the brain and improved A? clearance across the BBB to levels near that observed in untreated apoE3 animals. These studies suggest an isoform-specific relationship between apoE and MMP9. The hypothesis of this proposal is that apoE4 is less efficient than other apoE isoforms in regulating MMP9 function, which leads to increased lipoprotein receptor shedding and reduced A? elimination from the brain. The goal of this proposal is to elucidate the mechanism by which apoE regulates MMP9 function and influences lipoprotein receptor shedding and A? removal from the brain. The current proposal will examine several mechanisms to determine the relationship between apoE and MMP9. Aim1 will evaluate the impact of apoE genotype on MMP9 expression by examining MMP9 levels in isolated cerebrovasculature from AD mouse and human brains, each with different apoE genotypes. Aim2 will examine the influence of each apoE isoform on MMP9 disposition. These studies will investigate the effect of apoE on MMP9 cellular secretion, conversion of pro-MMP9 to active MMP9, and MMP9 activity. Aim3 will test the binding affinity between apoE and MMP9 and determine the effect of apoE on MMP9 binding to the lipoprotein receptor. In addition, the current proposal will examine the effect of MMP9-directed therapy on the AD phenotype in transgenic mice (Aim4). To our knowledge, no study has investigated the effect of MMP9 inhibition in an animal model of AD. It is anticipated these studies will provide rationale for the elevated A? brain burden and limited response to treatment that is observed in apoE4 transgenic animals and AD patients carrying the apoE4 allele, and pave the way for new therapeutic strategies in AD.